The effects of electrical stimulation of median nerve with a continuous theta burst pattern (EcTBS) on the spinal H-reflex were studied. Different intensities and durations of EcTBS were given to the median nerve to 11 healthy individuals. The amplitude ratio of the H-reflex to maximum M wave (H/M ratio), corticospinal excitability and inhibition measured using motor evoked potentials (MEPs), short-interval intracortical inhibition and facilitation (SICI/ICF), spinal reciprocal inhibition (RI), and postactivation depression (PAD) were measured before and after EcTBS. In result, the H/M ratio was reduced followed by EcTBS at 90% H-reflex threshold, and the effect lasted longer after 1200 pulses than after 600 pulses of EcTBS. In contrast, EcTBS at 110% threshold facilitated the H/M ratio, while at 80% threshold it had no effect. Maximum M wave, MEPs, SICI/ICF, RI, and PAD all remained unchanged after EcTBS. In conclusion, EcTBS produced lasting effects purely on the H-reflex, probably, through effects on postsynaptic plasticity. The effect of EcTBS depends on the intensity and duration of stimulation. EcTBS is beneficial to research on mechanisms of human plasticity. Moreover, its ability to modulate spinal excitability is expected to have therapeutic benefits on neurological disorders involving spinal cord dysfunction. Plasticity is an intrinsic reaction to adapt to environmental pressures, physiologic changes, and experiences . There aSpinal plasticity is important in the functional recovery of several neurological disorders. For instance, synaptic plasticity is known to occur simultaneously with axonal sprouting and cellular proliferation after spinal cord injuries (SCIs) and may contribute to improve motor function –8. AnothIn contrast, spinal plasticity may also cause neurological disorders. Spasticity, which is one of the most common sequelae of SCIs and strokes, may be the best example. Although spasticity involves complex changes in the spinal cord and muscles –15, it iSo far, only a few noninvasive protocols, including transcutaneous electrical nerve stimulation (TENS) , patternIn the present study, we developed a protocol for producing an inhibitory LTD-like effect in the spinal cord. Continuous theta burst stimulation (cTBS) based on repetitive TMS over M1 has shown its efficiency and efficacy in the suppression of cortical excitability through a LTD-like mechanism –31. We tEleven healthy subjects without history of neurological disorders were recruited. Eight of the subjects  underwent the complete experiments and 10 subjects participated in the experiment of spinal reciprocal inhibition. One subject, who did not have a consistent H-reflex in the forearm, was excluded. All participants signed their informed consent prior to the participation. The project protocol was approved by the Institutional Review Board of Chang Gung Memorial Hospital, Taiwan.Each subject was seated in a comfortable chair. EMGs were performed with 1 cm-diameter Ag/AgCl-plated surface electrodes placed 2 cm apart over the tested muscles in the dominant hand as instructed separated below in each experiment. The EMG of the flexor carpi radialis muscle , extensor digitorum communis muscle , and abductor pollicis brevis muscle  were recorded. The EMG was amplified and band-pass filtered (3 Hz to 2 kHz) by Digitimer D360 amplifiers . Signals were recorded at a sampling rate of 5 kHz and stored on the computer for later analysis by Signal software  through a power 1401 data acquisition interface . If the target muscle was not relaxed (as monitored with an EMG gain of ×5000) during the test, the data would be scrapped and the test redone.EcTBS was adapted from the theta burst protocol (TBS) of repetitive transcranial magnetic stimulation (rTMS)  and consμV, in a random order. A total of 6 trials given every 6–8 sec were recorded in each condition. The intensities that produced the 2 maximum H-reflexes were used for testing the effect of EcTBS afterwards. Mmax was also recorded from the same FCR muscle by using supramaximal stimulation. Then EcTBS was applied on the median nerve. Two maximum H-reflexes  and Mmax were recorded again at 0, 15, 30, 45, and 60 min after the end of cTBS EcTBS. The H/M ratio was calculated by dividing the peak-to-peak amplitude of H-reflex by the peak-to-peak amplitude of Mmax. Two H/M ratios were then averaged for analysis.The effects of EcTBS were firstly evaluated with the maximum H-reflex and Mmax. H-reflexes were recorded from FCR by stimulation of the median nerve. Electrical median nerve stimulation was performed through surface electrodes to stimulate the median nerve in the antecubital fossa with the duration of 1 ms. In the baseline condition, the H-reflex recruitment curve was recorded with the stimulus intensities at 90%, 100%, 120%, 140%, 160%, 180%, and 200% of H-reflex threshold, which was defined as the minimum stimulus current to the evoked-potential with an amplitude of 50 We started the experiments with EcTBS600 at 90% and 110% H-reflex threshold intensity and discovered a better inhibitory effect at an intensity of 90% (see below for the results). Since we aimed to find an optimal inhibitory protocol, we doubled the stimulus duration to have EcTBS1200 at 90% and 80% H-reflex threshold for the following study. We expected to achieve a better result by doubling the length of stimulation because studies of rTMS usually find that the effect lasts longer after longer periods of stimulation. In addition, 80% H-reflex threshold may have produced better inhibition than 90%, since there was better inhibition at 90% than at 110% threshold using EcTBS600.μV in more than five out of ten trials from the contralateral APB while the subject was maintaining a voluntary contraction of about 20% of the maximum using visual feedback. Subjects received the stimulation in a random order either of the test stimulus alone or of conditioning-test stimuli for a total of 12 trials per condition. The intertrial interval was 4.5–5.5 s. SICI/ICF was calculated as the ratio of the mean conditioned MEP over the mean unconditioned test MEP. The unconditioned test MEPs in SICI/ICF assessments were used to evaluate any change in the size of MEP.In this session, TMS was used to evaluate the effect of EcTBS on excitatory and inhibitory circuits in the motor cortex. TMS was given using a hand-held figure of eight coils with a mean loop diameter of 70 mm  connected to a Magstim Bistim2 machine . The coil was placed tangentially to the scalp over the “motor hot-spot” with the handle pointing backwards. The “motor hot-spot” was marked on the scalp and defined as the location where magnetic stimulation produced the largest MEP in the APB muscle. APB was selected as the target muscle because like FCR it is innervated by the median nerve but has a clearer MEP than FCR. Short-interval intracortical inhibition and intracortical facilitation (SICI/ICF) were assessed in the baseline condition and at 30 min after the EcTBS1200 at 90% H-reflex threshold. SICI/ICF were tested using a paired-pulse technique with a subthreshold conditioning stimulus followed by a suprathreshold test stimulus of TMS at interstimulus intervals (ISI) of 3, 7, and 10 ms . The conμs, and the intensity was adjusted to produce an M wave around 50 μV from EDC [In a separate experiment, RI in the same forearm was assessed. RI assesses the interaction between stimulation of the radial nerve and the H-reflex produced by stimulation of the median nerve . Electrifrom EDC –36. We rPAD was tested before and 30 min after spinal EcTBS1200 at 90% H-reflex threshold in the same forearm to understand the presynaptic mechanism of the spinal reflex in EcTBS. Electrical stimulation was performed via surface electrodes. Stimulation duration lasted for 1 ms, and the intensity of the stimuli was adjusted to evoke maximal H-reflexes from FCR in the baseline condition. Four conditions composed of two electrical median nerve stimuli at ISIs of 1, 3, 5, and 7 sec were given every 30 ± 10% sec in a random order for a total of six trials in each condition. PAD was calculated as the ratio of the conditioned (second) H-reflexes to the unconditioned (first) H-reflex at each trial and was averaged for each condition.Experiments were done in different days at least two weeks apart from each other in a random order.t-test (baseline and 30 min after EcTBS1200). For PAD, a two-way ANOVA with factors of TIME (before and 30 min after) and ISI  was performed to compare PAD before and after EcTBS. Furthermore, a one-way ANOVA on unconditioned baseline H-reflex and conditioned H-reflexes at ISI of 1, 3, 5, and 7 sec followed by post hoc tests was performed to test the effect of ISI on PAD. Differences were considered significant at a level of 5% or below.A two-way repeated measure ANOVA on values normalized to the baseline with factors of TIME  and PROTOCOL were applied to compare the effects on the H/M ratio between different EcTBS protocols. A one-way ANOVA analysis on nonnormalized values followed to test the effect of TIME  for the effect of the individual protocol. The results of MEP, SICI/ICF, and RI were analyzed by the paired F = 2.129, P = 0.127). The effect of EcTBS600 at different intensities was then compared on values normalized to the baseline  = 14.515, P = 0.007), for EcTBS600 at 110% H-reflex threshold facilitated the H/M ratio  = 6.394, P < 0.001), while EcTBS600 at 90% H-reflex threshold inhibited the H/M ratio  = 2.580, P = 0.043), mainly at 30 min after EcTBS. We then compared the effects of EcTBS1200 at 90% and 80% threshold and revealed an INTENSITY × TIME interaction  = 10.148, P = 0.015)  = 3.173, P = 0.018), while EcTBS1200 at 80% threshold failed to produce any effect  = 1.953, P = 0.110). Furthermore, we compared the effects of EcTBS600 and EcTBS1200 at the same intensity of 90% threshold. Two-way ANOVA showed a significant interaction between TIME × DURATION (EcTBS600 and EcTBS1200)  = 3.187, P = 0.028), indicating that EcTBS1200 produced a more profound inhibitory effect than EcTBS600. We further tested the amplitude of Mmax and found that none of the protocols of EcTBS changed Mmax .There was no significant difference between the baseline H/M ratio in all conditions of experiment 1 (baseline . A two-w= 0.015) . A follot-tests revealed that EcTBS1200 at 90% threshold did not alter the amplitudes of unconditioned MEP  , ICF at ISI of 10 ms , and SICI/ICF at intermediate ISI (7 ms)  (Paired = 0.917) , SICI at= 0.975) .t-tests revealed no significant difference at all phases after EcTBS1200 .The results of RI (before and 30 min after EcTBS1200) at all ISIs are shown in F = 6.976, P = 0.002) but no significant effect of TIME  = 0.668, P = 0.441) and no TIME × ISI interaction  = 1.307, P = 0.298). The results indicate that PAD was not changed by EcTBS. We therefore put pre- and post-EcTBS data together to evaluate the effect of ISI on PAD. A one-way ANOVA showed a significant effect of ISI indicating that the second H-reflex was significantly suppressed by the first H-reflex. The post hoc test confirmed that the second H-reflex was significantly suppressed at ISI of 1, 3, and 5 second(s) but not at 7 seconds .The results of PAD (before and 30 min after EcTBS1200) at all ISIs are shown in In our experiments, the H/M ratio was suppressed following EcTBS at 90% H-reflex threshold. Eighty seconds of EcTBS containing 1200 pulses suppressed the H/M ratio for 45 min or more, while 600 pulses of EcTBS produced a shorter-lasting inhibitory effect. Interestingly, increasing the stimulus intensity to 110% threshold did not enhance the suppressive effect but produced a facilitation instead. By contrast, 80% threshold produced no detectable effect on the H/M ratio. None of the protocols changed the size of Mmax. Moreover, spinal reciprocal inhibition, the size of MEP, and SICI/ICF remained unchanged at 30 min after EcTBS1200 at 90% threshold, when the H-reflex was most suppressed.EcTBS modifies the H/M ratio and leaves the amplitude of Mmax unchanged, suggesting that EcTBS mainly affects the circuit of the H-reflex. The H-reflex involves a monosynaptic loop, in which the afferent consists of Ia fibers from the muscle spindles and the efferent consists of alpha motor axons, within the spinal cord . The lacTo further clarify the mechanism of EcTBS at the spinal level we examined spinal reciprocal inhibition in the same nerve. There are three known phases of RI in the forearm. The first phase that occurs at ISI of 0 ms or so in the radial-medial stimulation is regulated by the glycinergic disynaptic inhibitory pathway, while the second phase that occurs at ISI of 10–20 ms includes the presynaptic Ia inhibitory pathway , 41. TheMoreover, we tested PAD to identify the location of the plasticity within the synapse of the H-reflex. Postactivation depression was thought to be due to the presynaptic mechanism involving a decreased probability of neurotransmitter release from previously active Ia-afferent terminal –46. No c2+ influx that tends to induce LTP [At first glance, we were surprised to see that EcTBS600 at 110% threshold facilitated the H-reflex. cTBS600 using rTMS generally shows an inhibitory effect over the cerebral cortex , althougduce LTP , 53. AltDifferent from EcTBS over M1, EcTBS600 at 90% threshold only produced a relatively short-lasting inhibitory effect around 30 min after the end of EcTBS, indicating that spinal circuits are less modifiable than those in the brain. It is reasonable since the brain needs to be more flexible than the spinal cord for, say, learning and memorizing. After doubling the length of stimulation, EcTBS1200 is capable of producing a more profound and consistent inhibitory effect for 45 min or more. This result implies that, at least within a certain range, the longer the stimulation lasts, the stronger and longer-lasting after-effect on the H-reflex it may produce. This is consistent with a previous report of TBS using rTMS . NeverthEcTBS modulates the H-reflex likely through a mechanism involving synaptic plasticity in the human spinal cord. The effect of EcTBS depends on its stimulus intensity. Intensities higher than the H-reflex threshold tend to facilitate the H-reflex, while intensities slightly below the threshold suppress H-reflexes. Moreover, a longer-lasting EcTBS induces a more consistent and longer-lasting after-effect than a shorter EcTBS. Hence, EcTBS1200 at 90% threshold is an efficient protocol to produce an inhibitory LTD-like effect in the human spinal cord and it can be used for exploring mechanisms of human plasticity, understanding the pathophysiology of diseases relevant to spinal plasticity and, hopefully, providing therapeutic benefits on spinal disorders in the future.


To investigate the association and magnitude of risk between JIA, its associated treatment and cancer development in Taiwanese children.Nationwide population-based 1:4 age- and gender-matched retrospective cohort study was designed using the National Health Insurance Research Database of Taiwan. A cohort of 2,892 children <16 years old with JIA was formed as well as a non-JIA cohort of 11,568 in year 2003 to 2005. They were followed up till a diagnosis of malignancy or up to 8 years until 2010. Relative risk (RR), incidence rate ratio (IRR), and adjusted hazard ratio (aHR) of developing malignancy were calculated.The female to male ratio was 0.79:1. There were 3 cases of incident cancer in the “MTX use, biologics-naïve” group, only 1 in the anti-TNF biologics-containing group and 29 in the “both MTX- and biologics-naïve” group, in comparison, there were 50 cases of cancer in the non-JIA comparator group. During a 16114.16 patient-years follow-up, the RR and IRR for developing a malignancy in both methotrexate- and anti-tumor necrosis factor (TNF) biologics-naïve JIA children were 2.75  and 3.21 (2.01 – 5.05), respectively. For leukemia, the IRR was 7.38 (2.50 – 22.75); lymphoma, 8.30 (1.23 – 69.79); and soft tissue sarcoma, 11.07 (0.84 – 326.4). The IRR of other cancers was 2.08 (1.11 – 3.71). The aHR on cancer risk was 3.14 (1.98 – 4.98) in methotrexate- and biologics-naïve group. There were no statistically significant increased risk in JIA patients treated with methotrexate and/or anti-TNF biologics.Compared with children without JIA, children with JIA have 3-fold increase of risk on malignancy in East Asia. Seemingly neither methotrexate nor anti-TNF biologics increases the risk further. Juvenile idiopathic arthritis (JIA) is a spectrum of several heterogeneous disorders manifested mainly as chronic inflammatory arthritis with no identifiable etiology with an onset before the age of 16 years. The older terms, juvenile rheumatoid arthritis (JRA) and juvenile chronic arthritis (JCA) have been replaced by the term juvenile idiopathic arthritis which is a more inclusive term than JRA .–20.18–20A recent detailed analysis of 100 cases of T-cell non-Hodgkin’s lymphoma (NHL) reported to the US FDA disclosed that 68% of the cases involved exposure to both a TNF-alpha inhibitor and an immunomodulator. Risk of T-cell NHL was higher with concomitant use of TNF-alpha inhibitor with thiopurines  and thiopurines alone  but not with TNF-alpha inhibitor use alone  . Many ofThe female to male ratio in our JIA cohort was 0.79: 1. This ratio echoes other prior studies from Asia that in contrast to Caucasian population having female preponderance which ranges from 2: 1 to 3: 1 in most published series, Asian population of JIA has a relative male predominance. The explanation for this sex ratio difference is fairly clear that ERA, an entity of predominantly male and older age of onset, constituted 42% of the JIA cohort in this study. Our previous study mentioned earlier in the paper found that the proportion of ERA was 37.4% in the Taiwanese Chinese cohort , 6, 9.In the JIA cohort, 15.5% ((344 + 105) / 2,892) of children required methotrexate for aggressive preservation of joint function and control of disease process. And only 3.9% of the entire JIA cohort were escalated to anti-TNF biologics therapy. In a published nationwide retrospective survey of 570 cases in Japan, 12.8% of their case series were treated with methotrexate [Our study has certain strengths. Because participation in the NHI program is mandatory and all residents of Taiwan can assess a paediatrician rheumatologist without needing a referral, the referral bias is essentially avoided. Loss to follow-up is virtually non-exist because of the country-wide NHI coverage for medical care allowing continuous tracking of an enrollee during relocation or self-transferal. With a large number of age- and gender-matched non-JIA comparators, the cumulative incidence rate of cancer in the control group is considered accurate.However, the findings of this study need to be interpreted in the context of certain potential limitations. First, many demographic variables were not available, such as family history of cancer, diet and environmental exposures. This is because individual identities are not available due to the de-identification of the individuals within the NHI databases. Second, the disease severity of JIA could not be obtained in this claims study. The risk of over-diagnosis resulted from solely using the ICD-9-CM codes for case identification is likely present due to inability to verify the diagnosis of JIA using the patients’ own medical records. We could not be 100% sure to exclude potentially exist undiagnosed JIA cases in the control group which may result in possible allocation bias. Finally, since this is not an incidence cohort for JIA and left censoring of disease and medication therapy cannot be excluded.The possibility of a protopathic bias was not completely ruled out although the study design had deployed an exclusion criteria to exclude subjects with pre-existing cancer traceable back to at least one whole year in the medical record by ICD-9-CM codes.This nationwide population-based retrospective cohort study of the association between JIA by their treatment allocation and cancer risk with up to 8 years follow-up reveals that this East Asian children population with JIA is associated with a 3-fold increased risk of malignancy (IRR = 3.21 and adjusted HR = 3.14) and there is no statistically significant increased risk in these patients with exposure to methotrexate and/or TNF inhibitors. The risk for other solid tumors was statistically significantly increased by 2-fold.JLH and KWY are professors of pediatric rheumatology and experts in juvenile idiopathic arthritis. VCK is a medical oncologist and medical informatician who is also teaching evidence-based medicine to junior medical staffs. JTH and CWC are experts in information engineering and big data management.


Background. Osteopontin (OPN) has been proved to be associated with allergic airway inflammation. However, the roles of OPN and its regulation in childhood allergic rhinitis (AR) are poorly understood. Objective. This study aims to evaluate the expression of OPN and miR-181a in children with AR and their association with Th1/Th2 immune response. Methods. Children who suffered from AR were included along with control subjects. Serum was collected to examine the level of OPN and Th1/Th2 cytokines by enzyme-linked immunosorbent assay (ELISA) and the level of miR-181a by quantitative polymerase chain reaction (qPCR). Results. Children with AR had significantly higher serum levels of OPN and lower serum levels of miR-181a than healthy controls. Furthermore, serum levels of OPN were positively correlated with Th2 cytokine and negatively correlated with Th1 cytokine. On the contrary, miR-181a level had a negative correlation with IL-4/IL-5 and positive correlation with IFN-γ/IL-12. More importantly, there was also significant negative correlation between OPN and miR-181a. Conclusion. The OPN protein and miR-181a levels may serve as predictors of disease severity in childhood AR and appear to be promising targets for modulating AR.  Allergic rhinitis (AR) is a common chronic respiratory disease of the upper airway which is characteristically presented with rhinorrhea, sneezing, and congestion . The oveOsteopontin (OPN) is an extracellular matrix protein and cytokine that could be produced by the airway epithelial cells and inflammatory cells around airways. It is a multifunctional protein in allergy and asthma, an immune modulator that has been recognized as a key regulator in immunoglobulin (Ig) E-mediated and Th2-skewed allergic response . Also, OMicroRNAs (miRNAs) are small non-protein-coding RNAs that function as posttranscriptional regulators of target genes expression by suppressing translation or through mRNA degradation . MicroarTwenty-five children with AR were recruited at the Department of Otolaryngology, Guangzhou Women and Children's Medical Center, from January 2015 to October 2015. The diagnosis of AR was made on history, clinical examination, skin prick test, and specific IgE measurement according to the Allergic Rhinitis and Its Impact on Asthma (ARIA) guideline (2010) . Twenty Venous blood samples were obtained into Vacuette tubes between 6 am and 8 am after an overnight fast. After centrifuging at 1000 g for 15 min at 4°C, serum samples were separated and stored at −80°C until assay.−ΔΔCt method and normalized to RNA U6 controls.For miR-181a, total RNA from each serum sample was reverse transcribed into cDNA using the TaqMan microRNA reverse transcription kit and TaqMan microRNA assays (Invitrogen), as per manufacturer's instructions. The results were calculated with the 2γ, IL-5, IL-12, and IL-4 were measured in duplicate using ELISA kits  according to the manufacturer's instructions. The detection limits of the assays were as follows: OPN (312.5 pg/mL), IFN-γ (12.5 pg/mL), IL-4 (1.56 pg/mL), IL-5 (7.8 pg/mL), and IL-12 (2.5 pg/mL). Eosinophil cationic protein (ECP) was detected by Unicap system.The serum levels of OPN, IFN-t-test. A P < 0.05 was set as a significant criterion.The data are presented as mean ± SD. The differences between groups were determined by Student's We enrolled 25 children with AR and 20 normal controls for the study. Anthropometric parameters of the AR children and controls were listed in r = −0.58, P < 0.05; r = −0.59, P < 0.05).Concomitant OPN and miR-181a levels in serum were evaluated. As shown in γ/IL-12 . The results also revealed a significant inverse correlation between serum miR-181a level and TNSS .To identify if OPN and miR-181a have relationship with the severity of pediatric AR patients, the TNSS of the children was assessed. As shown in There is limited literature regarding the roles of OPN and miRNAs in airway allergic inflammation and most relates to asthma and adult population. In this study, we demonstrated that the serum level of OPN was elevated and miR-181a was decreased in AR children compared to that in normal controls. OPN level was positively correlated with Th2 cytokine IL-4 and total nasal symptom score, whereas miR-181a level had a negative correlation with them. Our key finding was that the levels of OPN and miR-181a had an inverse correlation in the serum of children with AR.OPN is expressed in a variety of cells, including bronchial epithelial cells, airway and vascular smooth muscle cells, myofibroblasts, and inflammatory cells like T-lymphocytes and mast cells . The eleThe biological functions of OPN can be profoundly influenced by posttranslational modifications . AccumulA number of studies have recognized the important roles of OPN in Th1-associated immunological diseases such as Crohn's disease  and tubeγ. The findings are supported by previous observations demonstrating that serum OPN concentration was correlated with blood serum ECP and IL-5 levels which were also typical Th2 cytokines in adult AR [γ. Based on the above analysis, it seems plausible that miRNA-181a acted on OPN to modulate the expression of pro- and anti-inflammatory cytokines in the setting of childhood AR.Our present study in pediatric AR demonstrated a positive correlation between OPN and IL-4, a representative cytokine of Th2-mediated allergic responses, and negative correlation with Th1 cytokine IFN-adult AR . Furtheradult AR . MeanwhiWe emphasize that the altered systemic OPN and miR-181a are related to the modulation of Th2-mediated allergic immune response. Therefore, it is reasonable to ask whether both of them contribute to the severity of AR in clinical scenario. In the present study, serum OPN showed a significant positive relationship and miR-181a had an inverse correlation with total nasal symptom scores in pediatric AR patients. These findings suggest that OPN plays a crucial role in driving the progression of childhood AR and miR-181a is more likely to have opposing effect. Nonetheless, the question of whether decreased miR-181a mediated posttranscriptional silencing is a potential mechanism for the upregulation of OPN needs further investigation.In summary, children with AR showed a marked increase in serum level of OPN but a decrease in miR-181a level. Our findings may be beneficial for designing a potential strategy for the optimal prevention and management of AR children.


Gossypium hirsutum) is one of the most important worldwide crops it provides natural high-quality fiber for the industrial production and everyday use. Next-generation sequencing is a powerful method to identify single nucleotide polymorphism markers on a large scale for the construction of a high-density genetic map for quantitative trait loci mapping.Upland Cotton  with a recombinant inbred line population using single nucleotide polymorphism markers developed by specific locus amplified fragment sequencing. We also identified quantitative trait loci of boll weight across 11 environments and identified candidate genes within the quantitative trait loci confidence intervals. The results of this research would provide useful information for the next-step work including fine mapping, gene functional analysis, pyramiding breeding of functional genes as well as marker-assisted selection.This research reported the first high-density genetic map for Upland Cotton (The online version of this article (doi:10.1186/s12870-016-0741-4) contains supplementary material, which is available to authorized users. Gossypium hirsutum L., 2n = 52) is widely grown because it provides superior natural fiber for the textile industry and daily life [Upland cotton  technique can be used to detect large quantities of SNP markers in the whole genome . There au et al.  also conIn this study, a recombinant inbred line (RIL) population, containing 196 individuals was developed from an intra-specific cross between two upland cotton 0–153 and sGK9708. We attempted to use this population to construct an intra-specific HDGM of upland cotton, to identify QTLs and possibly, the candidate genes correlated to cotton boll weight. Finally, a total 5521 SNP markers were successfully applied to genotype these 196 RILs along with parents and an intra-specific HDGM was thus constructed. This map was used to identify QTLs for cotton boll weight across 11 environments.6:8 recombinant inbred lines (RIL) population of upland cotton with 196 individuals was developed from a cross between homozygous cultivars 0–153 and sGK9708. Cultivar 0–153 harbored superior fiber quality traits while sGK9708 was derived from CRI41 which maintained high yield potential and wide adaptability. The details of the development of RILs have been already described by Sun et al. [The intra-specific Fn et al. . Addition et al. .Thirty normally opened bolls within five to eight fruiting branches and one to three fruiting nodes were sampled in annually September. The total seed-cotton of the 30 bolls was weighted and average boll weight was calculated accordingly. One-way ANOVA was used to test the significance of the differences in boll weight between two parents. Additionally, EXCEL 2010 was used to create the descriptive statistics including the mean value, standard deviation, skewness and kurtosis of the boll weight across the whole population.Gossypium hirsutum [in silico simulation of the number of markers generated by various endonuclease combinations. The SLAF library was constructed based on the SLAF pilot experiment in accordance with the predesigned scheme and eventually two endonucleases combination of HaeIII and SspI  was applied to the genomic DNA digestion in our RIL population. The details of SLAF-seq strategy was described by Zhang et al. [The leaves of the parents and the RIL population were sampled in July and stored at −70 °C. The genomic DNA was extracted using the TaKaRa MiniBEST Plant Genomic DNA Extraction kit  and SLAF-seq strategy with some modifications was utilized in the library construction. Briefly, the reference genome of hirsutum , 19 was g et al. .Gossypium hirsutum [SLAF markers were identified and genotyped with procedures described by Sun et al.  and Zhanhirsutum  sequencehirsutum . Sequenchirsutum . SNP lochirsutum . The repThe procedure of all polymorphic SLAF loci genotyping was described by Sun et al.  and ZhanLinkage map was constructed based on the procedure detailed by Zhang et al.  and the p < 0.05) were still maintained to construct the HDGM, the region on the map with more than three consecutive adjacent loci that showed significant (0.001 < P < 0.05) segregation distortion was defined as a segregation distortion region (SDR) [As the distortedly segregated markers showing significance between 0.001 and 0.05 (0.001 < on (SDR) . The sizAll the sequences of SNP markers that were constructed in the linkage map were aligned back to the physical sequence of the upland cotton genome through local Basic Local Alignment Search Tool (BLAST) to confirm their physical positions in the genome. Software CIRCOS 0.66 was used to compare the collinearity of markers based on their genetic positions and physical positions. The recombination hotspot (RH) was estimated based on the recombination rate of markers. If the value that the genetic distance between adjacent markers was divided by was higher than 20 cM/Megabase, the region between the two adjacent markers was regarded as RH .P < 0.05, n = 1000. LOD score values between 2.0 and permutation test LOD threshold were used to declare suggestive QTL. Positive additive effect means that the favorable alleles come from the 0–153 parent while negative additive effect means that the favorable alleles come from sGk9708. QTLs were named and the common QTLs were identified as described by Sun et al. [Windows QTL Cartographer 2.5  was usedn et al. .p values were considered as the enriched pathways. The candidate genes were also categorized based on their products through eukaryotic orthologous groups (KOG) database analysis.The markers flanking the confidence intervals of the QTLs which can be detected in at least three environments were selected to identify the candidate genes. The sequences of these markers were aligned back to the physical sequence of upland cotton genome database . Based op-value was 0.002, suggesting that significant differences of boll weight were found between the two parents. The descriptive statistical analysis results of the RIL population and parents across 11 environments were shown in Table The one-way ANOVA result showed the After SLAF library construction and sequencing, 87.89 GB of data containing 443.56 M pair-end reads was generated with each read of 80 bp in length. Among them, 82.24 % of the bases were of high quality with Q20  and guanine-cytosine (GC) content was 34.47 %. The SLAFs numbers of 0–153 and sGK9708 were 53,123 and 53,238, and their correspondent sequencing depths were 78.66 and 102.13 respectively. The coverage of both parents was 35 %. In the RIL population, the number of SLAFs ranged from 32,261 to 53,104 and the average number of SLAFs was 50,487. The average sequencing depth was 14.50, and the average coverage was 33.37 %  were filtered and the remaining 5521 markers, including the ones that showed significant segregation distortion between 0.05 and 0.001 (0.001 < P < 0.05) were used to construct the final genetic map  segregation distortion. These segregation distortion markers (SDMs) were located in the chromosomes with an uneven distribution in each. Among the 1225 SDMs, 579 of them were located in the A subgenome of upland cotton whereas 646 of them were located in the D subgenome of upland cotton. Chromosome 14 had the largest number of SDMs and accounted for the highest percentage of SDMs of all the mapped markers. The number of SDMs on c14 was 238 and accounted for 58.33 % of the total markers mapped on it. Chromosome 22 had the smallest number of SDMs (four). Chromosome 4 had 4.7 % SDMs, the lowest overall percentage. In total, 93 SDRs were defined in all the chromosomes, with 44 of them located in the A subgenome of upland cotton and the other 49 located in the D subgenome of upland cotton. Chromosome 14 had the most SDR number, 18 SDRs, while chromosomes 4, 8, 17, 20, 22, and 24 had no SDR . QTL qBW-chr13-4, detected in six environments, was located within the marker interval of CRI-SNP8313-CRI-SNP-8346, and explained 4.58–6.06 % of the observed PV. QTLs qBW-chr01-1 and qBW-chr25-5, both of which were detected in five environments, were located within the marker intervals of CRI-SNP147-CRI-SNP168 and CRI-SNP10564-CRI-SNP10569, and explained 4.81–7.83 % and 4.29–10.76 % of the observed PV respectively. QTLs qBW-chr02-3, qBW-chr07-1, qBW-chr07-6, qBW-chr09-6 and qBW-chr25-7, all of which were detected in four environments, located within the marker intervals of CRI-SNP506-CRI-SNP519, CRI-SNP-5634-CRI-SNP5581, CRI-SNP5454-CRI-SNP-5438, CRI-SNP6432-CRI-SNP6455 and CRI-SNP10592-CRI-SNP10615, and explained 5.62–6.41, 4.95–8.89, 5.35–10.89, 5.01–10.31 and 7.58–7.80 % of the observed PV respectively. QTLs qBW-chr03-1, qBW-chr05-10, qBW-chr07-4, qBW-chr16-4, qBW-chr22-3, qBW-chr23-5 and qBW-chr25-6, all of which were detected in three environments, were located within the marker intervals of CRI-SNP-1241-CRI-SNP-1231, CRI-SNP-2294-CRI-SNP-2279, CRI-SNP-5497-CRI-SNP5472, CRI-SNP12560-CRI-SNP12270, CRI-SNP10330-CRI-SNP10341, CRI-SNP13838-CRI-SNP13865 and CRI-SNP10569-CRI-SNP10571, and explained 4.56–9.00, 5.64–7.45, 6.92–8.45, 4.15–5.03, 6.64–8.80, 4.26–5.26 and 4.82–11.85 % of the observed PV respectively . Sixteen of them were regarded as stable QTLs as they could be detected in at least three environments. In the confidence intervals of these stable QTLs, qBW-chr02-3 which has no candidate gene, the confidence intervals of all the remaining QTLs have candidate genes. The confidence intervals of qBW-chr07-4 and qBW-chr25-6 harbored only one candidate gene whereas the confidence interval of qBW-chr23-5 harbored 65 genes . When restriction endonuclease(s) were applied to the genome digestion and selected properly, the fragments generated by next-step sequencing would be a better representation of the genome. In the previous studies, usually a few common restriction endonucleases such as  species . Typical species –32. The  species –33. This species , 33. The species –14. Compossypium , 34, 35 hirsutum and G. barbadense were applied to the genetic map construction, the coverage and resolution of the map could be greatly improved [hirsutum and G. barbadense may not show polymorphism within the cultivars of G. hirsutum. SNP markers could improve the coverage and resolution of the genetic map efficiently. Wang et al. [Gossypium hirsutum genome. In the initial steps of marker development through SLAF-seq, the quantities of SLAFs developed were about the same sizes in the different chromosomes. After several steps of screenings, the remaining numbers of SNPs for map construction varied greatly among the chromosomes, and the reduced number of remaining SNPs contributed to the shortness of some chromosomes. The collinearity comparison between the genetic map and the physical one validates the reliability of the constructed map. However, a better understanding of the genetic structure of these chromosomes might need an integrative analysis.In previous studies, most of the genetic maps of cotton were based SSR markers. The low polymorphic rate of the SSR markers makes the SSR marker based maps unable to harbor a sufficient number of markers with a comparative poor coverage of the genome and low resolution. In most cases, these maps have large gaps, and sometimes the gap divides the chromosome into two or more linkage groups , 36, 37.improved –40. HoweG. hirsutum and a G. barbadense to construct a genetic map and identified 10 QTLs on eight chromosomes . The confidence intervals of these QTLs also harbored two or three markers and spanned distances from 2 to 30 cM. In our study, we identified the QTL of the boll weight in 25 chromosomes except chromosome 8. Among them 16 QTLs were detected in at least three environments and were present on 11 chromosomes . The confidence intervals of these QTLs harbored from two to 26 markers ranging from 0.7 to 13.9 cM. This implies that our results of QTL identification are more concise and accurate than previous studies and could be useful for future research looking at gene identification or cloning from these QTLs, or even breeding practices using MAS.Previous QTL studies were primarily focused on the fiber quality traits , 2, 40, Among the 16 stable QTLs that can be detected in at least three environments, eight had positive additive effects whereas the other eight had negative additive effects. This indicates that both the higher boll weight value parent sGK9708 and lower boll weight value parent 0–153 could contribute positive additive QTLs to increase the boll weight. This could be a possible factor behind the difference in the boll weight trait between the parents 0–153 and sGK9708. Theoretically, the greater the difference of one trait between the two parents, the higher the possibility that the positive additive effect of the QTLs would come from one parent. The RIL population was constructed primarily based on differences in fiber quality traits especially fiber strength between the parents 0–153 and sGK9708, therefore, the difference of fiber strength is larger than that of any other traits between 0–153 and sGK9708. In Sun’s report , seven QGh_A07G1188, Gh_A07G1197and Gh_D09G1606 had a relation to signal transduction mechanisms. Two genes, Gh_A05G1210 and Gh_D04G1531 were related the function posttranslational modification, protein turnover, and chaperones. Two genes, Gh_A07G1187 and Gh_A13G0858, had the translation function, ribosomal structure, and biogenesis, though this function basket did not harbor a large number of candidate genes. As the posttranslational modification, protein turnover and ribosomal structure were relative to the protein synthesis, it is probable that the genes correlated to this function contribute to the boll weight trait.Among all 340 candidate genes being annotated in at least one channel of KOG, KEGG, and GO, some might be related to the boll weight trait. In KOG analysis, there were 21 function baskets. The posttranslational modification function, protein turnover, chaperones and signal transduction mechanisms harbored the largest number of candidate genes. Among the 44 genes located closest to the markers of genetic position, three genes Gh_A13G0858 has a relationship to the ribosome, whereas genes Gh_A13G0392 and Gh_D06G0187 have a relationship to the plant hormone signal transduction. As the ribosome has a relationship to protein synthesis and some plant hormones such as auxin and gibberellin, these genes could contribute to the plant growth and eventually to the boll weight trait, particularly the gene Gh_A13G0858.In KEEG analysis, the first three pathways which harbored the largest number of genes were plant hormone signal transduction, and protein processing in endoplasmic reticulum and ribosome, harboring six genes, four genes and four genes respectively. Of these 14 genes, three were located at the nearest positions of the markers, genetic position of which had the highest LOD values in the QTL mapping analysis. The gene Although these genes were located the nearest position of the markers, genetic position of which had the highest LOD values in the QTL mapping analysis, but there still lacks direct evidence to prove that the function of these genes was correlated to the boll weight trait.Gossypium hirsutum) with a RIL population using SNP markers developed by SLAF-seq. The HDGM had a total number of 5521 markers and a total distance of 3259.37 cM with an average marker interval of 0.78 cM. There were no gaps greater than 10 cM.We also identified QTLs of boll weight trait across 11 environments and identified candidate genes. Totally, 146 QTLs of boll weight was identified and 16 of them were detected in at least three environments with a stable QTL. Three hundred forty-four candidate genes were identified in the confidence intervals of stable QTLs and 44 of them were located in the nearest positions of the markers. The result of this research would provide information for the next phase of research such as fine mapping, gene functional analysis, pyramiding breeding and marker-assisted selection (MAS) as well.This research reported the first HDGM of Upland Cotton (The data sets supporting the results of this article are included within the article and its additional files.


HPV transmission dynamics have rarely been studied in the general population, especially in China. We followed the genital HPV infection status of both partners in 874 couples aged 25-65 years from rural China for up to 7 bi-annual visits during 2009-2013. The positive HPV concordance and transmission rate for partners in a couple were evaluated and relevant risk factors were assessed. The concordance of any, oncogenic, and non-oncogenic HPV was 15.52%, 16.18% and 10.41%, respectively. Male-to-female transmission rate was 7.11, 12.13 and 4.77/1000 person months for any, oncogenic and non-oncogenic HPV respectively. The female-to-male transmission rate was 5.56, 2.37, and 17.01/1000 person months for any, oncogenic and non-oncogenic HPV respectively. The risk of male-to-female transmission was significantly higher than that of female-to-male transmission for oncogenic types. However, for non-oncogenic types, the risk of male-to-female transmission was significantly lower than that of female-to-male transmission. Younger couples, persistent infection with HPV, higher numbers of sexual partners and higher frequency of sexual intercourse were positively associated with HPV transmission in couples. Our results indicate that men in rural China play a more important role than men in western populations as a source of cervical oncogenic HPV infection in women. HPV infection has been demonstrated to be the causative agent of cervical cancer in women, and it also plays an important role in anogenital and oropharyngeal cancers236789676789In this study we followed genital HPV infection status in 874 couples aged 25 to 65 for up to 7 bi-annual visits from 2009 to 2013 by making use of an ongoing population-based cohort study in rural Anyang, Chinand cross-section investigation of the AECCS in 2009-2010. The eligibility criteria for couples included in the current study were as follows: 1) being married and permanent resident in the target villages; 2) age 25-65 years; 3) female partner had an intact uterus and was not pregnant during the study period; 4) no history of cancer, cardiovascular disease or mental disorder; 5) no history of infection with HBV, HCV, or HIV; and 6) willingness to cooperate with the follow up and provide informed consent. All eligible couples were enrolled from 2009 to 2010 and were assessed bi-annually, for up to 7 visits for husbands and up to 6 visits for wives. Centralized examinations were conducted, and all couples were sampled within a period of two weeks at each follow-up visit. Study visits for male and female partners were conducted at the same visit.In 2007–2009 a population-based esophageal cancer cohort study (AECCS) was initiated in rural Anyang, ChinaFor males, exfoliated cells from the penile shaft, glans penis (prepuce was not included), coronal sulcus, and scrotum were collected by an experienced doctor using saline-soaked swabs at each evaluationResearch protocols and materials were approved by the Institutional Review Board of the Peking University School of Oncology, China. All methods were carried out according to the approved guidelines. All participants in this study provided informed written consent.Specimen DNA was extracted using the Biomek 3000 workstation . The β-globin gene was first evaluated by PCR, and only β-globin positive specimens were subjected to HPV evaluation using a pair of SPF1/GP6+ primers131517For concordance analysis, only positive concordance of HPV on the basis of a type-specific principle was evaluated. Any couple meeting the conditions: a) both male and female provided ≥2 valid HPV results within the study period; b) either partner had at least 1 HPV positive visit, was included in the analysis. HPV concordance was defined as the proportion of co-positive couples for a specific HPV type out of all HPV exposed couples. For groups of genotypes , concordance was defined as the proportion of couples co-positive for at least one of any type HPV, 14 oncogenic HPVs and 32 non-oncogenic HPVs. The Chi-square test was performed for specific HPV types to determine whether the observed concordant events significantly exceeded the expected valueAnalysis of male-to-female and female-to-male transmission was conducted separately under a type-specific principle. The infection was treated as the calculating unit and multiple infections in one participant were counted as separate events. In this study, we observed that the transmission event was far less likely to occur after the infection in the partner in whom HPV infection originated had been cleared (data not shown). In view of this fact, we employed an “HPV transmission window” to define the transmission event and calculate the person-time at risk of HPV transmission in couples. The “transmission window” opens at the time of the visit when one partner in a couple is found to be positive for a specific type of HPV for the first time, while his/her partner has been negative for the corresponding HPV type from enrollment to the time of that visit. The window closes at the earliest occurrence of any one of the following: a) the adjacent visit subsequent to the last positive visit of this infection in the HPV index partner, b) a type-specific transmission event occurs, c) the follow-up of the at-risk partner right censored. An HPV transmission event was defined as newly acquired type-specific infection in the at-risk partner within the transmission window. A couple was included in the transmission analysis if the at-risk partner had at least 2 consecutive visits within the “HPV transmission window”. Transmission rates were then calculated by dividing the number of transmission events by the total number of person months of transmission windows, and 95% CIs were estimated based on Poisson distributionFactors for HPV transmission evaluated in the current study included average age, average lifetime number of sexual partners and sexual frequency per week of study couples, as well as the level of education, type of job and presence of persistent HPV infection in a given couple. Persistent HPV infection was defined as ≥3 consecutive positive visits for the HPV-exposed partner in this study. Factors associated with HPV concordance were assessed using a logistic regression model, in which the Generalized Estimating Equation (GEE) was used to take into consideration of within subject correlationAll statistical analyses were performed using STATA version 11.2 . Tests were two sided, and had a significance level of 0.05.In this study, 93.4% and 98.3% of genital exfoliated cell specimens from men and women were positive for the human beta-globin gene and were thus considered adequate for subsequent HPV testing. A total of 874 couples provided at least one valid HPV positive result for both partners, among whom HPV positivity was found in 749 of 4480 visits (16.7%) in men, and 345 of 4215 visits (8.2%) in women during the study period. The proportion of multiple infection was 8.5% in men (64/749) and 8.7% in women (30/345). Among these 874 couples, 406 (46.5%) HPV exposed couples provided at least 2 valid specimens from both partners and were included in the concordance evaluation. The median follow-up time was 33.6 months for males (IQR 18.8-38.0) and 30.4 months for females (IQR 23.8-31.9). For transmission analysis, 296 HPV exposed couples in whom HPV transmission had potential to occur within the defined transmission window were included with a median follow-up time of 35.7 months for males (IQR 19.4-38.3) and 30.5 months for females (IQR 25.1-32.2).p < 0.001) reported having multiple sexual partner. Around half of these couples  reported having sexual intercourse ≤1 each week. Persistent HPV infections were detected more frequently in males . No appreciable differences were observed in couples included in or not included in the concordance analysis and the transmission analysis, except that greater numbers of lifetime sexual partners were observed in couples included in the concordance analysis as compared with excluded couples (p = 0.013).p = 0.054, for oncogenic vs. non-oncogenic). Specifically, concordance for HPV-16, HPV-18, HPV-11 and HPV-6 was 12.17%, 5.45%, 22.22% and 42.86%, respectively  in male-to-female transmission. The female-to-male transmission rates for any HPV type, oncogenic, non-oncogenic HPV and HPV-16/18/11/6 infection were 5.56, 2.37, 17.01 and 2.73 per 1000 PM, respectively. The transmission rate of oncogenic types was significantly lower than non-oncogenic types (p = 0.012) in female-to-male transmission.Male-to-female transmission rates for any HPV type, oncogenic, non-oncogenic HPV and HPV-16/18/11/6 infection were 7.11, 12.13, 4.77 and 10.48 per 1000 PM respectively as shown in M-to-F vs. F-to-M = 4.89, 95%CI: 1.36-17.55; p = 0.015), but the male-to-female transmission rate was significantly lower than the female-to-male transmission rate in non-oncogenic HPV types . For vaccination types, the risk of male-to-female transmission was higher than that of female-to-male transmission, but this difference did not reach a significant level   .For factor analyses, a significantly lower risk of concordant infection for both any and oncogenic HPV was observed in older couples aged 46-65 years, as compared with couples aged 25-45. Persistent HPV infection was consistently associated with an increased risk of both HPV concordance and transmission. Couples who reported having >1 lifetime sexual partner had a significantly elevated risk of HPV concordance in any and oncogenic HPV, and also had a significantly elevated risk of any type of HPV transmission. Risk of both HPV concordance and transmission in non-oncogenic types was significantly increased in couples who reported having 2 episodes of sexual intercourse per week as compared to couples who reported having sexual intercourse ≤1 per week .In this study, we investigated the positive concordance and transmission rate of genital HPV infection among 874 couples aged 25-65 from northern rural China with population-based sampling. We found that observed concordance in the majority of HPV types was significantly higher than expected. The risk of male-to-female transmission was around 4 times higher than that of female-to-male transmission for oncogenic HPV, and conversely, the male-to-female transmission rate of non-oncogenic HPV was 73% lower than that of female-to-male transmission.et al.et al.et al., the mean number of lifetime sexual partners was 11.0 for men and 5.3 for womenet al. reported that the proportion of >5 lifetime sexual partners was 64.4% for men and 59.4% for womenet al. also reported that the median number of lifetime sexual partners was 12.0 for men and 7.0 for womenet al. found the proportion of ≥3 lifetime sexual partners was 74.7% and 73.7% in men and womenUntil now only 5 prospective studies have investigated HPV transmission in heterosexual couples. All these studies67891926p = 0.003). In the 5 published studies we evaluated for comparison, only Burchell et al. reported the proportion of “persistent” infection after 4 months, and estimates found of 59.5% for men and 60.6% for women were without significant gender difference2930Another possible explanation was the similar prevalence but higher proportion of persistent infection in oncogenic HPVs in males as compared to females observed in our study  younger couples are more sexually active, 2) older couples have longer periods of HPV exposure, and antibodies generated by previous infections may protect against reinfection by identical HPV types192930Two limitations of this study should be noted. First, although we have investigated over 800 couples for as many as 7 visits, statistical power is still limited when analyzing specific types due to the low prevalence and transmission incidence of genital HPV infection in this population. Larger studies with greater numbers of cycles of evaluation are still needed to validate our conclusions. Secondly, as couples in the current study were not asked to avoid sexual intercourse for 48 hours before each visit, superficial virus deposited by sexual partners during recent intercourse may have confounded results. However this would likely not significantly impact our conclusions in view of the fact most couples reported having ≤1 episodes of sexual intercourse per week.The results of our study indicate men in rural China play a much more important role than men in other populations as a source of genital oncogenic HPV infection. While anti-HPV intervention programs are already being planned for women to decrease the burden of HPV-related disease in this population, this further argues for the necessity of such programs in young and sexually active male adults. This is the first population-based prospective study employing a rigorous periodic sampling method to assess genital HPV transmission in a Chinese population. As no national HPV and cervical cancer screening program has been established in rural China, and in view of the fact prophylactic vaccines are still under clinical trial in China pending government approval, our findings may be of significance in targeting a high risk population for development of cost-effective vaccination-based cervical cancer and HPV screening programs.How to cite this article: Liu, M. et al. Transmission of genital human papillomavirus infection in couples: a population-based cohort study in rural China. Sci. Rep.5, 10986; doi: 10.1038/srep10986 (2015).


Studies concerning the association between circulating resistin and mortality risk have reported, so far, conflicting results.To investigate the association between resistin and both all-cause and cardiovascular (CV) mortality risk by 1) analyzing data from the Gargano Heart Study (GHS) prospective design ; 2) performing meta-analyses of all published studies addressing the above mentioned associations.MEDLINE and Web of Science search of studies reporting hazard ratios (HR) of circulating resistin for all-cause or CV mortality.Performed independently by two investigators, using a standardized data extraction sheet.In GHS, adjusted HRs per one standard deviation (SD) increment in resistin concentration were 1.28 (95% CI: 1.07-1.54) and 1.32 (95% CI: 1.06-1.64) for all-cause and CV mortality, respectively. The meta-analyses included 7 studies  for all-cause mortality and 6 studies  for CV mortality. Pooled HRs per one SD increment in resistin levels were 1.21  and 1.05  for all-cause and CV mortality, respectively. At meta-regression analyses, study mean age explained 9.9% of all-cause mortality studies heterogeneity. After adjusting for age, HR for all-cause mortality was 1.24 (95% CI: 1.06-1.45).Our results provide evidence for an association between circulating resistin and mortality risk among high-risk patients as are those with diabetes and coronary artery disease. Several studies indicate that resistin, a 12.5 kDa cysteine-rich pro-inflammatory adipokine, is a major promoter of atherosclerosis and related cardiovascular (CV) disease , includiSince CV disease is the first cause of mortality worldwide, resistin becomes a natural candidate to investigate as a possible mortality risk factor. In fact, conflicting results have been reported on this subject –16.To deeper understand the role of resistin on mortality rate, we firstly added new evidences by trying to replicate our previous finding  in a secThis study comprises 368 patients with type 2 diabetes mellitus (T2DM) (ADA 2003 criteria) and coronary artery disease (CAD) who were consecutively recruited at the Endocrine Unit of IRCCS “Casa Sollievo della Sofferenza” in San Giovanni Rotondo  from 2001 to 2008, as recently described , 17. AllClinical data were obtained from a standardized interview and examination. Smoking habits and history of hypertension, dyslipidemia and MI as well as glucose-lowering treatment were also recorded at time of examination. Data regarding medications were confirmed by review of medical records.Serum resistin was measured in 359 (98%) participants by a commercial ELISA  at the Research Unit of Diabetes and Endocrine Diseases in San Giovanni Rotondo, as previously described .The study was approved by the Institutional Ethic Committee IRCCS “Casa Sollievo della Sofferenza”, San Giovanni Rotondo. All participants gave written consent.The present meta-analysis follows the Preferred Reporting Items for Systematic Review and Meta-analyses (PRISMA) statements . Risks were reported as HRs along with 95% CI.Dose-response meta-analysis followed a two-step process and was performed for all-cause and CV mortalities outcomes, separately.Step 1: We considered those studies in which HRs were reported with respect to a study-specific categorical reference dose of serum resistin concentrations (i.e. tertiles or quartiles) , 11–13: Step 2: We considered those studies in which HRs were reported with respect to one increment of resistin SD  , 14–16 aStep 3: Given that resistin measurement was carried out in different specimens (i.e. serum or plasma), by different laboratories using different assays, we firstly converted the scaled measures with respect to one increment of study-specific SD, and then all study-specific slopes obtained from Step 1 and Step 2 were combined and their summary statistics were estimated. Statistical heterogeneity among studies was assessed using the Cochran Q-test and heterogeneity hold for p-values less than 0.10 . Study-sTo evaluate consistency and stability of HRs estimates found from meta-analysis and meta-regression, sensitivity analyses were carried out, by re-running all analyses after excluding one study at one time.Due to the small number of studies (fewer than ten) and the presence of substantial between-study heterogeneity, following Sterne et al.  and IoanForest plots were shown, where a square was plotted for each study whose center projection on the underlying scale corresponded to the study-specific HR. The area of the square was proportional to the inverse of the variance of the logHR and thus gives a measure of the amount of statistical information available from that particular estimate. A diamond was used to plot the summary HRs, the center of which represents the HR; the extremes of the summary HRs show the 95% CI.Bubble plot of study-specific logHRs is shown against the chosen study-level covariate which explained the most proportion of heterogeneity. The size of each bubble was inversely proportional to the standard error of specific logHR.Two-sided p-values<0.05 were considered for statistical significance. Statistical analysis were performed using SAS Software, Release 9.3  and R 2.15 (package: metafor).Clinical features of study participants are summarized in During follow-up , 81 all-cause and 58 CV deaths occurred, corresponding to an annual incidence rate of 4.2 and 3.0 events per 100 person-years, respectively.-5 and HR = 1.34, 95%CI: 1.14–1.57, p = 3x10-4, respectively; Each SD (i.e. 6.6 ng/ml) increment of serum resistin was associated with an increased risk of both all-cause and CV mortality . Sensitivity and meta-regression analyses were then performed . At sensitivity analysis, HR estimates per each unitary increment of SD in resistin levels turned out to be stable (ranging from HR = 1.13 to HR = 1.26) and quite consistent with the HR obtained from all studies. At meta-regression analysis, among all study-level covariates which were available from all studies , study mThe number of subjects from each study ranged from 150 to 1888 with a mean follow-up ranging from 1.0 to 5.5 years .With the only exception of our current study, none of the previous ones reported a significant association with CV mortality , 15, 16,The pooled HR per one increment in SD of resistin concentration for CV mortality was 1.05 , using the fixed-effects model .No significant heterogeneity across study results was observed (Q-test p for heterogeneity = 0.20), so that no meta-regression analysis was carried out.Data obtained from the GHS-prospective design confirm our previous observation that serum resistin is a predictor of all-cause mortality  and exteSince heterogeneity was observed across studies for all-cause mortality, meta-regression analysis was employed to get more reliable pooled HRs estimates. Although the observed results have to be taken with great caution because of the limited number of available studies, heterogeneity was partly explained by study mean age (i.e. stronger association in studies with older subjects).Though our meta-regression allows speculate that age acts as a modulator of the resistin effect on all-cause mortality, only studies with individual patient data may say a last word about this hypothesis. Unfortunately, no studies addressing this specific issue are available.Finally, we observed a potential risk for publication bias; however, given the small number of studies (fewer than ten) and the presence of between-study heterogeneity, we believed unnecessary to address it in accordance with previous recommendations , 24.We have to recognize some study limitations, as follows. Our meta-analyses have been conducted in high-risk patients ; so, it is unknown whether our finding applies also to “healthy” individuals with low risk profile. In addition, most studies included in the meta-analyses were carried out in patients from European Countries, thus making impossible to generalize our findings to population of different ancestry and/or environmental background.In conclusion, our results provide evidence for an association between higher circulating resistin levels and increased mortality risk. Further studied are needed to verify whether or not including resistin in risk engines which are already validated –28 improS1 PRISMA Checklist(PDF)Click here for additional data file.S1 Supporting Information(DOCX)Click here for additional data file.


These findings confirm that chronic fisetin treatment exerts antinociceptive effect on thermal hyperalgesia in neuropathic mice, with spinal serotonergic system (coupled with 5-HT7) being critically involved. Of special benefit, fisetin attenuated co-morbidly behavioral symptoms of depression and anxiety  evoked by neuropathic pain.Fisetin, a natural flavonoid, has been shown in our previous studies to exert antidepressant-like effect. As antidepressant drugs are clinically used to treat chronic neuropathic pain, this work aimed to investigate the potential antinociceptive efficacies of fisetin against neuropathic pain and explore mechanism(s). We subjected mice to chronic constriction injury (CCI) by loosely ligating the sciatic nerves, and Hargreaves test or von Frey test was used to assess thermal hyperalgesia or mechanical allodynia, respectively. Chronic fisetin treatment  ameliorated thermal hyperalgesia  in CCI mice, concomitant with escalated levels of spinal monoamines and suppressed monoamine oxidase (MAO)-A activity. The antihyperalgesic action of fisetin was abolished by chemical depletion of spinal serotonin (5-HT) but potentiated by co-treatment with 5-HTP, a precursor of 5-HT. Moreover, intraperitoneal (i.p.) or intrathecal (i.t.) co-treatment with 5-HT Neuropathic pain is usually an enduring and devastating condition resulting from damage to nociceptive pathways in the peripheral or central nervous system35Fisetin . Experimental behavioral tests were performed in a soundproof and air-regulated room and were done in blind respect to surgical procedure and drug treatment. All experiments and animal handing were in accordance with the European Communities Council Directive of 24 November 1986 (86/609/EEC) and approved by the Ningbo University Animal Committee. The authors tried all efforts to minimize the number of animals used and their suffering.Adult male C57BL/6J mice  were used in this study. Animals were group-housed (4–6 per cage) with food and water available The chronic constriction injury (CCI) model was used to induce neuropathic pain as delineated by Bennett and Xie1A receptor antagonist), ritanserin (5-HT2A/2C receptor antagonist), ondansetron (5-HT3 receptor antagonist) and SB-258719 (5-HT7 receptor antagonist). The doses of these drugs were selected on the basis of our previous studies  began 15 days after the surgical procedure (CCI or sham-operation), when the CCI mice exhibited obvious nociceptive hypersensitivity i.e. thermal (heat) hyperalgesia and mechanical allodynia. For chronic treatment, the sham and CCI mice received two oral administrations , intracerebroventricular (i.c.v.) or intraplantar (i.pl.) injection of 5-HTIntrathecal injections were done as described by Hylden and WilcoxThe procedures for intracerebroventricular injections were based on the methods described by Haley and McCormickIn a separate set of experiments, intraplantar injections were performed under ether anesthesia. A 29-gauge injection needle punctured the ipsilateral plantar skin and was placed in the subcutaneous space just proximal to the footpads. A volume of 10 μl was used for i.pl. injection.p-chlorophenylalanine , suspended in 0.5% gum acacia/physiological saline, was administered i.p. for five consecutive days at a dose of 300 mg/kg/dayAfter treatment with fisetin or vehicle for 3 weeks, pharmaceutical manipulations were performed in mice to deplete spinal NA or 5-HT. For chemical denervation of spinal noradrenergic transmission, the mice were injected intrathecally with catecholaminergic neurotoxin 6-OHDA (20 μg per mouse), which was dissolved in 5 μl of 0.9% saline containing ascorbic acid (100 μg/ml)17st fisetin treatment on the day, shown in 20Thermal hyperalgesia and mechanical allodynia were used as outcome measures of pain-related behaviors in mice and were evaluated by Hargreaves test and von Frey test, respectively. The depression-like behaviors in mice were measured by forced swim test (FST) and novelty suppressed feeding test (NSFT), while the anxiety-like behaviors were assayed by light-dark test (LDT) and NSFT. In FST, NSFT and LDT, the animals were tested only once, i.e. the different time points of tests were done on independent sets of animals. In Hargreaves test and von Frey test, the animals were tested consecutively in a time-course fashion. To investigate the acute actions of fisetin, the behavior tests were performed at different time points  after a single fisetin administration on day 15. In the experiment settings for chronic fisetin treatment, the behavioral tests were done 1 hour before the morning fisetin treatment  hyperalgesia was assessed based on the Hargreaves procedureVon Frey test: Mechanical allodynia was tested using a series of von Frey monofilaments (Stoelting USA) and results  were expressed as grams. Mice were placed in transparent Perspex cubicles with an elevated wire mesh bottom, and allowed to habituate for at least 20 min before testing. The filaments were applied perpendicularly to the plantar surface of hind paw in a series of ascending forces . Each filament was tested five times per paw, and the mechanical threshold was defined as the minimal force that caused at least three withdraws observed out of five consecutive trialsForced swim test (FST): FST was conducted according to the method established by Porsolt et alNovelty suppressed feeding test (NSFT): NSFT reveals both depression and anxiety-like properties in rodents. The testing apparatus consists of a 40 × 40 × 30 cm plastic box with floor covered with 1 cm sawdust. Food was removed from the home cage 24 h before the test. At the beginning of testing, a pellet of food was placed on a paper in the center of the box. A mouse was then placed in a corner of the box and the latency to first contact or eat the pellet was recorded within a 5-min period. This procedure elicits a conflict between the drive to eat the food and the fear of venturing in the center of the boxLight-dark test (LDT): The apparatus consists of light and dark boxes (25 × 18 × 20 cm each) connected by a dark tunnel (9 × 7 × 6 cm). The lit compartment was brightly illuminated (1500 lux). Mice were placed in the dark compartment in the beginning of the test and the time spent in the lit compartment was recorded during 5 min.Locomotor activity test: The assessment of locomotor activity was performed in mice according to our previous studiesFollowing 3 weeks fisetin or vehicle treatment, the mice were decapitated and the lumbar enlargements (approximately 1 cm) of their spinal cords were rapidly dissected out and collected. The contents of monoamines  and their metabolites (5-HIAA and DOPAC) were measured using high-performance liquid chromatography (HPLC) with electrochemical detection  or one-way ANOVA. For multifactor-ANOVA, for example, the surgery (sham or CCI) and the treatment (vehicle or drug administration) were done as between-group factors. When needed, the time of measurement (time-course data) was done as a within-subject factor. The Duncan test was used for post hoc comparisons. For one-way ANOVA, Student-Newman-Keuls test was used for multiple comparisons, followed by Student's test to evaluate the difference between two groups at the same time. Differences with The neuropathy induced by peripheral nerve injury (CCI) produced in mice ipsolateral  thermal hyperalgesia and mechanical allodynia, which were persistent and maintained throughout the experiment . In cont7,284 = 12.6, p < 0.01; After 3 weeks treatment, fisetin markedly attenuated in CCI mice the pronounced thermal hyperalgesia  pathway. The escalated spinal 5-HT contents and depressed ratio of 5-HIAA/5-HT in CCI mice, following fisetin treatment, indicate the alteration of monoamine metabolism may be more likely responsible for the antinociceptive actions of fisetin. The monoamine in CNS is metabolized by two isoforms of MAO (MAO-A and MAO-B), with MAO-A preferentially metabolizing serotonin and NA while MAO-B dealing with NA and dopamine. We then evaluated the effect of chronic fisetin treatment on the MAO-A and MAO-B activities in mouse spinal cord. As shown in 6,231 = 10.4, p < 0.01; 24To determine whether the monoamine system is causally implicated in the antihyperalgesia by fisetin, we investigated the effects of depleting spinal 5-HT or NA on the antihyperalgesic effect of fisetin. It has been reported that a single intrathecal (i.t.) injection of 6-OHDA or consecutive intraperitoneal (i.p.) injections of PCPA reduced spinal NA or 5-HT content, respectively246,228 = 9.1, p < 0.01), pointing to a potentiating effect when fisetin was co-treated with 5-HTP. These results validate the serotonergic mechanism underlying the antihyperalgesic action of fisetin.To further support the crucial role for serotonergic system in the antihyperalgesic action of fisetin. We examined whether fisetin antihyperalgesia can be influenced by co-treatment with 5-HTP, a precursor of serotonin. As shown in 1A receptor antagonist WAY-100635 , 5-HT2A/C receptor antagonist ritanserin , 5-HT3 receptor antagonist ondansetron  and 5-HT7 receptor antagonist SB-258719 . In CCI mice, the antihyperalgesic action of fisetin was abolished by repeated co-administration of the selective 5-HT7 receptor antagonist SB-258719  and tested its effect on the antihyperalgesic effect of fisetin. As shown in 2,116 = 32.6, p < 0.01) in CCI mice, without influencing the measures in sham mice. Whereas, i.c.v. (5 μg in 2.5 μl) or i.pl. (1.5 μg in 10 μl) injection of SB-258719 did not modify the nociceptive responses to thermal stimuli in both sham and neuropathic mice.To further investigate 5-HTIt has been reported that rodents (mice and rat) rendered neuropathic by peripheral injury manifested co-morbid depressive and anxiety-like behaviors14153,38 = 12.1, p < 0.01; 3,39 = 16.6, p < 0.01; 3,40 = 9.2, p < 0.01; After 3 weeks treatment, fisetin dose-dependently attenuated in CCI mice the co-morbidly behavioral phenotypes of depression and anxiety. (a) In FST, fisetin  dose-dependently decreased  the prolonged immobility time , acute fisetin treatment exhibited positive activities in sham-operated mice . But thiAs shown in 7 receptors. In addition, persistent mononeuropathy elicited in mice co-morbidly behavioral symptoms of depression and anxiety and these phenotypes can also be palliated by chronic treatment with fisetin.In the present study, we show that fisetin, when chronically and orally administered in mice with mononeuropathy, exerted antihyperalgesic effects. This action is modality and time dependent since it is sensitive to thermal (heat) stimuli but not to mechanical one, and is present following chronic rather than acute fisetin treatment. Mechanistically, the antihyperalgesia by fisetin may depend on the activation of descending serotonergic system, which is coupled with spinal 5-HTThe CCI model from Bennett and Xie1415th day following CCI-lesion when the pain-related behaviors had been established, thus implying its potential utility in the development of novel and clinically relevant drugs for the treatment of neuropathic pain. Accordingly, it would be intriguing to know whether fisetin can prevent the development of chronic pain by preemptive administration. Secondly, its antinociceptive action is modality and time dependent, inasmuch as it works in fighting thermal hyperalgesia but is actually not responsive to mechanical hypersensitivity, and this antihyperalgesia was observed following repeated but not single dose fisetin regimen. Thirdly, it is notable to point out that the antihyperalgesia by fisetin shown in the present study should be a genuine one, since the nociceptive tests were performed on the next morning following the last fisetin administration and thus the antihyperalgesic action is present for at least one day. This point is important and indicates that the antihyperalgesia provided by fiseitn is persistent but not transient. Lastly, our results delineates the spinal site of fisetin antinociception, which is similar to previous reports concerning action site of some antidepressant drugs20The main finding of the present study is the ability of fisetin to induce relief of thermal (heat) hyperalgesia in neuropathic mice following 3 weeks of oral treatment. To our best knowledge, it is the fist report to demonstrate the antinociceptive activity of fisetin and its antinociceptive action incorporates several characteristic aspects. Firstly, the antihyperalgesia of fisetin is a reversing action (not a prophylactic one) as the treatment started on the 15The descending monoamine pathway, especially that involving noradrenergic and serotonergic transmission, is a major component of the endogenous pain modulatory system7 receptor antagonist SB-258719 completely abolished the antihyperalgesic action of fisetin, but it is not the case for other 5-HT receptor antagonists . These observations strongly argue for the key role of 5-HT7 receptors underlying fisetin antihyperalgesia and are congruent with recent studies reporting the engagement of 5-HT7 mechanism in the initiation and maintenance of neuropathic pain197 receptors to spinal but not supraspinal or peripheral level, as i.t., but not i.c.v. or i.pl., injection of SB-258719 counteracted the antihyperalgesic action of fisetin. This is supported by immunocytochemical studies showing that 5-HT7 receptors are localized in the superficial layers of the spinal dorsal horn, a key region for pain modulation7 receptors. Of note, the inhibition of antihyperalgesic action of fisetin by blockade of 5-HT7 receptors requires a repeated co-administration process, which is reminiscent, as for modality and time-course, of the relapse of hyperalgesia after fisetin withdrawal. This temporal delay suggests that the escalation of serotonergic tone may be a primary or upstream target for fisetin action.To further investigate the pharmacological mechanisms behind fisetin action, we tested different 5-HT receptor antagonists to identify which subtypes of 5-HT receptors are responsible for the antihyperalgesic effects of fisetin. Our results unequivocally show co-administration of the 5-HT1, while fisetin and resveratrol coupled with 5-HT7). This is interesting and worthy of further investigations. In light of the present results and those obtained in previous reports197 selectivity of fisetin and resveratrol? Are other mechanisms such as inhibition of 5-HT re-uptake and increase of 5-HT release involved in the antinociceptive actions exerted by these compounds?Recently, we also screened the antinociceptive bioactivities of several other naturally occurring compounds such as curcuminPatients with chronic neuropathic pain often suffer from affective disorders like depression and anxiety14157), we propose that the enhanced spinal 5-HT tone (coupled to 5-HT7 receptors) may exclusively account for the mechanism behind fisetin antihyperalgesia. Over time, the mice with mononeuropathy developed behavioral symptoms of depression and anxiety. Our findings also confirm that chronic fisetin treatment can significantly improve aspects of these co-morbidly behavioral endophenotypes.In conclusion, the present study demonstrates that fisetin, when administered orally and chronically in neuropathic mice, can correct their thermal (heat) hyperalgesia, indicating a potential use of this naturally occurring molecule in the clinical setting of chronic neuropathic pain. Although further studies should be undertaken to fully elucidate the precise mechanisms for fisetin action (especially the predominant receptor selectivity of 5-HTX.Z. designed this study. X.Z., C.W., W.G.C., Q. M. and W.H.Z. performed the experiments. X.Z., C.W. and W.H.Z. analyzed the data. X.Z. wrote the paper. All authors read and approved the paper.


Leishmania is unclear.After the onset of HAART, some HIV-infected individuals under treatment present a exacerbated inflammation in response to a latent or a previously treated opportunistic pathogen termed immune reconstitution inflammatory syndrome (IRIS). Few reports of tegumentary leishmaniasis have been described in association with IRIS. Moreover, the immunopathogenesis of IRIS in association with Leishmania antigens. After receiving anti-leishmanial treatment, the individual’s specific Th1 immune response was restored.The present study reports on a 29-year-old HIV-infected individual who developed mucocutaneous leishmaniasis associated with immune reconstitution inflammatory syndrome (IRIS) five months following highly active antiretroviral therapy (HAART). Severe lesions resulted in the partial destruction of the nasal septum, with improvement observed 15 days after treatment with Amphotericin B and corticosteroids. The immune response of this patient was evaluated before and after the lesions healed. IRIS was diagnosed in association with high levels of TNF-α and IL-6. Decreased production of IFN-γ and a low IFN-γ/IL-10 ratio were also observed in response to The results suggest that the production of inflammatory cytokines by unstimulated T-lymphocytes could contribute to occurrence of leishmaniasis associated with IRIS. Highly active antiretroviral therapy (HAART) has significantly benefited the majority of HIV-infected individuals. HAART results in a decrease in the plasma HIV-viral load and a partial recovery of CD4 + T-lymphocytes . As a coMycobacterium avium complex, Mycobacterium tuberculosis, cytomegalovirus or herpes zoster [Leishmania antigens.Most cases of IRIS are associated with the s zoster ,5. Few cs zoster -8. FurthLeishmania antigen (SLA),[+ and CD8+ monoclonal antibodies conjugated with phycoerythrin (PE) and allophycocyanin (APC). Cell acquisition was performed using a FACSAria Flow-Cytometer  and subsequently analyzed by Flowjo™ software . The cell division index (DI) was used to quantify the proliferation intensity of T-cell subsets (DI = 0.06 for CD4+ and 0.09 for CD8+ T-cells.) The frequencies of CD4+ and CD8+ T-cells producing intracellular cytokines were quantified using flow cytometry. PBMCs were cultured in the presence of SLA, PHA or culture medium for 18 h. Heat-inactivated human AB serum, brefeldin A and monensin were added to all cultures in the final four hours. Next, PBMCs were stained with anti-CD4-fluorescein isothiocyanate (FITC) and anti-CD8-APC, then permeabilized with PBS-BSA-Saponin 0.2% and incubated with anti-INF-γ-PE, anti-TNF-α-PE, and anti-IL-10-PE . Plasma cytokine levels were quantified using the BD Cytometric Bead Array (CBA) Human Th1/Th2 Cytokine Kit II .A case of mucocutaneous leishmaniasis in association with IRIS in an HIV-infected individual was recorded in 2009 at the Professor Edgar Santos University Hospital (HUPES), located in Salvador, Bahia–Brazil. The HUPES Institutional Research Review Board approved the present case report and informed written consent was obtained from the patient. Blood samples for immunological assessments were collected prior to and immediately after (the following day) the course of Amphotericin B and corticosteroid treatment. Peripheral blood mononuclear cells (PBMCs) were isolated by passage over a Ficoll-Hypaque gradient . PBMCs were labeled with 1.5 μM of carboxyfluorescein succinimidyl ester dye  and cultured for five days in the presence of either 10 μg/mL of soluble + T-cell count was 160 cells/mm3 and viral load was 92,479 copies/mL. In May 2009, he presented with ulcerative lesions on his face in association with nasal obstruction. At this point, his CD4+ T-cell count was 516 cells/mm3 with an undetectable HIV viral load. The lesions subsequently progressed, resulting in severe inflammation characterized by a pronounced swelling of the lips, nasal and mentum regions. He was admitted to Hospital Professor Edgard Santos (HUPES) in August 2009 . At this time, his CD4Leishmania spp. were found in both skin and lymph node biopsies. The skin test for Leishmania was positive (15 mm) and indirect ELISA for soluble L. brasiliensis antigens was positive, while ELISA for rK39 was negative. IgG anti-L. braziliensis levels were measured using indirect ELISA for soluble L. braziliensis antigens. The specie determination was further confirmed using a serial real-time quantitative PCR assay system, as described by Weirather, JL, 2011 [A skin biopsy revealed the presence of granulomatous lesions. Caseous necrosis was identified by a cervical lymph node biopsy. Amastigote forms of The patient was then diagnosed with mucocutaneous leishmaniasis as a manifestation of IRIS and HAART was discontinued. He was subsequently treated with 80 mg/day of Prednisolone and 1 mg/Kg/day of Amphotericin B from August to September 2009. After 15 days of treatment, improvement in the mucocutaneous lesions and in the patient’s cervical lymphadenopathy was observed  and CD8+ T-cell subsets (DI: 0.2)  is supportive of an IRIS diagnosis. Moreover, the initial clinical presentation of mucosal leishmaniasis, which appeared five months after HAART initiation, progressed to an intense inflammatory response with partial destruction of the nasal septum in less than four months.Our results suggest that the mucosal damage resulting from mucocutaneous leishmaniasis as a manifestation of IRIS in this patient was correlated with an unspecific inflammatory milieu. During the course of IRIS, CD4Leishmania, while his antigen-specific T-cell proliferation was undetectable. This discrepancy could be explained by the dynamic of immune restoration following HAART. The restoration of antigen-specific CD4+ T-cell responses in vitro is mostly correlated with CD4+ memory T-cell reconstitution; whereas the improvement of delayed type hypersensitivity is associated with the suppression of viraemia [+ T-lymphocytes for this patient, during IRIS or after healing of lesions. In addition, an impairment of in vitro proliferative response to Leishmania antigens could be linked to the activation and exhaustion of immune system found during IRIS [During IRIS, the patient had a positive skin test for viraemia . Howevering IRIS . Yet, inLeishmania antigens with respect to the development of skin and mucosal lesions cannot be excluded, since a Leishmania-specific response was not evaluated in situ. A specific recruitment of CD4+ and CD8+ T-cells to the ulcerative lesion is described in patients with both cutaneous and mucosal leishmaniasis [Leishmania antigens in peripheral blood is associated with intense recruitment of Leishmania-specific T-cells to the lesions, which is classically found in HIV-uninfected patients with disseminated leishmaniasis, yet cytokine production in tegumentary lesions was observed to be similar to that found in patients with mucocutaneous and cutaneous leishmaniasis [+ and CD8+ T-cells persist in treated lesions, the number of circulating antigen-specific CD4+ and CD8+ T-cells increases in peripheral blood [Leishmania antigens during IRIS might be due to sequestration of T-cells inside lesions. After healing, these cells were redistributed to peripheral blood.In the present case, the participation of a specific response to maniasis ,17. Specmaniasis . Indeed,maniasis . Followial blood ,20. Thus+ T-cells specific for pathogens other than Leishmania. These cells recirculate from lymphoid organs into the peripheral blood stream during the first two months of HAART [The elevated cytokine production observed in non-stimulated cells may be a consequence of activated memory CD4of HAART ,21. Moreof HAART .in vitro, but not in his plasma. This finding may be due to the fact that post-treatment cytokine quantification was performed in the plasma by a single measurement just one day following the conclusion of treatment. It is possible that subsequent measurements would have found decreases in TNF-α in the plasma.High levels of IL-6 and TNF-α have been previously described in patients with other infectious diseases in association with IRIS -25. AddiLeishmania-specific CD4+ T-lymphocytes was not detected during the course of IRIS [Furthermore, the nonspecific activation of the immune system could be a consequence of an impaired regulatory T-cell response or a decrease in the proportion of these cells, since IL-10 produced by  of IRIS . The pro of IRIS ,30.+ T-cells following HAART. No convincing evidence has been presented in regard to whether CD4+ T-cells specific to opportunistic pathogens are deregulated during IRIS, much less if these cells are involved in the pathogenesis of IRIS. Moreover, the literature contains no reports that directly link CD4+ T-cells specific to Leishmania antigens with clinical manifestations of leishmaniasis in association with IRIS. However, several reports have shown a recovery of CD4+ T-cells specific to Mycobacterium tuberculosis, Mycobacterium avium complex and Cryptococcal neoformans in patients who developed IRIS in association with these infections [M. tuberculosis-specific CD4+ T-cells and tuberculosis in association with IRIS [IRIS is characterized by an intense inflammatory reaction, leading to tissue destruction concurrent with an increase in the number of CD4fections ,29,31. I+ T-cells are restored following HAART, these cells may intensely stimulate macrophages and possibly other innate immune cells that produce large amounts of proinflammatory cytokines, resulting in inflammation and tissue destruction [It has been suggested that the absence of T-cells, such as occurs in the course of AIDS, may lead to the growth of intracellular pathogens inside macrophages that never become fully activated, and thus exert no effector function. When antigen-specific CD4truction .Leishmania-associated IRIS occurred concurrently with the production of proinflammatory cytokines by unstimulated T-lymphocytes, which is supported by the absence of a specific host immune response against Leishmania antigens in the peripheral blood of this patient.In summary, the results presented herein suggest that Written informed consent was obtained from the patient for publication of this case report and any accompanying images. A copy of the written consent is available for review by the Editor of this journal.